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A Koltermann

Publications and source records attributed to A Koltermann.

8 recordsLinked to original sources
PubMed2001

Dual-color fluorescence cross-correlation spectroscopy for monitoring the kinetics of enzyme-catalyzed reactions.

Dual-color fluorescence correlation spectroscopy is a biophysical technique that enables precise and sensitive analyzes of molecular interactions. It is unique in its ability to analyze reactions in real time at nanomolar substrate concentrations and below, especially when applied to the monitoring of enzyme-catalyzed reactions. Furthermore, it offers a wide range of accessible reactions, restricted only by the prerequisite that a chemical bond or a physical interaction between two spectrally distinguishable fluorophores is established or broken. Recently, the optical setup of dual-color fluorescence correlation spectroscopy has been extended toward two-photon excitation, resulting in several advantages compared with standard excitation, such as lower fluorescence background, an even larger spectrum of potential fluorescence dyes to be used, as well as a more stable and simplified optical setup. So far, the method has been successfully employed to analyze the kinetics of nucleic acid and peptide modifications catalyzed by nucleases, polymerases, and proteases.

DNA↗
PubMed2000

Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis.

Confocal fluorescence correlation spectroscopy as a time-averaging fluctuation analysis combining maximum sensitivity with high statistical confidence has proved to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solutions, and in living cells. To probe the interaction of different molecular species for a detailed understanding of biologically relevant mechanisms, crosscorrelation studies on dual or multiple fluorophore assays with spectrally distinct excitation and emission are particularly promising. Despite the considerable improvement of detection specificity provided by fluorescence crosscorrelation analysis, few applications have so far been reported, presumably because of the practical challenges of properly aligning and controlling the stability of the experimental setup. In this work, we demonstrate that two-photon excitation combined with dual-color fluorescence correlation spectroscopy can be the key to simplifying simultaneous investigations of multiple fluorescent species significantly on a single-molecule scale. Two-photon excitation allows accession of common fluorophores of largely distinct emission by the same excitation wavelength, because differences in selection rules and vibronic coupling can induce considerable shifts between the one-photon and two-photon excitation spectra. The concept of dual-color two-photon fluorescence crosscorrelation analysis is introduced and experimentally demonstrated with an established assay probing the selective cleavage of dual-labeled DNA substrates by restriction endonuclease EcoRI.

Calibration↗
PubMed1999

Confocal fluorescence coincidence analysis: an approach to ultra high-throughput screening.

Fluorescence-based assay technologies play an increasing role in high-throughput screening. They can be classified into different categories: fluorescence polarization, time-resolved fluorescence, fluorescence resonance energy transfer, and fluorescence correlation spectroscopy. In this work we present an alternative analytical technique for high-throughput screening, which we call confocal fluorescence coincidence analysis. Confocal fluorescence coincidence analysis extracts fluorescence fluctuations that occur coincidently in two different spectral ranges from a tiny observation volume of below 1 fl. This procedure makes it possible to monitor whether an association between molecular fragments that are labeled with different fluorophores is established or broken. Therefore, it provides access to the characterization of a variety of cleavage and ligation reactions in biochemistry. Confocal fluorescence coincidence analysis is a very sensitive and ultrafast technique with readout times of 100 ms and below. This feature is demonstrated by means of a homogeneous assay for restriction endonuclease EcoRI. The presented achievements break ground for throughput rates as high as 10(6) samples per day with using only small amounts of sample substance and therefore constitute a solid base for screening applications in drug discovery and evolutionary biotechnology.

Base Sequence↗
PubMed1998

Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy.

A method for sensitively monitoring enzyme kinetics and activities by using dual-color fluorescence cross-correlation spectroscopy is described. This universal method enables the development of highly sensitive and precise assays for real-time kinetic analyses of any catalyzed cleavage or addition reaction, where a chemical linkage is formed or cleaved through an enzyme's action between two fluorophores that can be discriminated spectrally. In this work, a homogeneous assay with restriction endonuclease EcoRI and a 66-bp double-stranded DNA containing the GAATTC recognition site and fluorophores at each 5' end is described. The enzyme activity can be quantified down to the low picomolar range (>1.6 pM) where the rate constants are linearly dependent on the enzyme concentrations over two orders of magnitude. Furthermore, the reactions were monitored on-line at various initial substrate concentrations in the nanomolar range, and the reaction rates were clearly represented by the Michaelis-Menten equation with a KM of 14 +/- 1 nM and a kcat of 4.6 +/- 0.2 min-1. In addition to kinetic studies and activity determinations, it is proposed that enzyme assays based on the dual-color fluorescence cross-correlation spectroscopy will be very useful for high-throughput screening and evolutionary biotechnology.

Biochemistry↗
PubMed1998

Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: high throughput screening for enzyme activity.

Dual-color fluorescence cross-correlation spectroscopy (dual-color FCS) has previously been shown to be a suitable tool not only for binding but also for catalytic rate studies. In this work, its application as a rapid method for high-throughput screening (HTS) and evolutionary biotechnology is described. This application is called RAPID FCS (rapid assay processing by integration of dual-color FCS) and does not depend on the characterization of diffusion parameters that is the prerequisite for conventional fluorescence correlation spectroscopy. Dual-color FCS parameters were optimized to achieve the shortest analysis times. A simulated HTS with homogeneous assays for different restriction endonucleases (EcoRI, BamHI, SspI, and HindIII) achieved precise yes-or-no decisions within analysis times of about 1 s per sample. RAPID FCS combines these short analysis times with the development of fast and flexible assays resulting in sensitive, homogeneous fluorescence-based assays, where a chemical linkage between different fluorophores is either cleaved or formed, or where differently labeled molecules interact by noncovalent binding. In principle, assay volumes can be reduced to submicroliters without decreasing the signal strength, making RAPID FCS an ideal tool for ultra-HTS when combined with nanotechnology. RAPID FCS can accurately probe 10(4) to 10(5) samples per day, and possibly more. In addition, this method has the potential to be an efficient tool for selection strategies in evolutionary biotechnology, where rare and specific binding or catalytic properties have to be screened in large numbers of samples.

Biochemistry↗
PubMed1997

Principles and methods of evolutionary biotechnology.

Evolutionary biotechnology applies the principles of molecular evolution to biotechnology, leading to novel techniques for the creation of biomolecules with a great variety of functions for technical and medical purposes. Several basic principles for the application of evolutionary strategies can be derived from a comprehensive theory of molecular evolution. Prerequisites for evolutionary biotechnology are summarized with respect to the different classes of biomolecules and a few, selected applications are described in detail. Concepts for the technical implementation of evolutionary strategies are presented which allow automatized, high throughput processes.

Biotechnology↗
PubMed1997

Production of human interleukin-8 expressed in Escherichia coli: from a laboratory scale for in vitro tests via a technical scale for animal studies to a process scale for a GMP-compatible production.

An Escherichia coli K 12 strain has been constructed for efficient expression of recombinant biologically active human IL-8 (Interleukin-8). The development of a fermentation and purification process from the laboratory scale (cells from 15 l fermentation broth) to a production scale (cells from 200 l fermentation broth) is described. Material obtained from the laboratory scale was used for initial in vitro studies and for the development of a biological assay. An upscale purification process starting from 80 l fermentation broth resulted in larger amounts of IL-8 needed for preclinical studies. This process includes a fully automated control of the initial affinity chromatography step. Finally, a production process which differed markedly from the small-scale processes was tailor-made for GMP conformity and economic considerations. It consists of a cell disruption step followed by two crossflow diafiltrations with different molecular weight cut offs and filtration rates, one cation exchange chromatography and a final dialysis step. In order to enhance the overall yield of biologically active IL-8, conditions for a resolubilisation of insoluble IL-8 present in the remaining pellet after cell disruption were worked out.

Bioreactors↗